DNA Technology Manipulating DNA for our benefit Recombinant

DNA Technology: Manipulating DNA for our benefit! • • Recombinant DNA Cloning Gel Electrophoresis PCR

Technology #1: Recombinant DNA Genes from different sources are combined. • Ex. Frost resistance gene from potatoes put into strawberry DNA or resistance gene from bacteria into corn. Terms to know: • Vector -Carries target DNA into a living cell. – Example: Plasmid-Circular piece of bacterial DNA • Donor Gene – The target gene that scientists want to use from an organism. • Restriction Enzyme – Enzyme that can cut the strong bonds of DNA.

Plasmids do not contain genome DNA. They contain extra genes for reproduction and ab resistance.

Restriction enzymes cut the DNA in a specific spot. Sticky ends are open pieces of DNA that attach and seal to the new gene with ligase. Sticky Ends Plasmid

Figure 20. 3 -2 Restriction site 5 3 GAATTC CTTAAG DNA 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. 5 5 3 G CTTAA 5 Sticky 3 3 end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 5 3 3 AATTC G 3 5 G AATT C C TTAA G 5 3 5 5 3 AATTC G G CTTAA 3 5 3 5 G AATT C C TTAA G 5 3 One possible combination 3 5

Creating Recombinant DNA 1. Target gene is cut of donor genome using restriction enzymes. 2. Foreign DNA is inserted into a plasmid (cut with the same enzyme). 3. The recombinant DNA is inserted into a bacterial cell. © 2011 Pearson Education, Inc.

4. Reproduction in the bacterial cell results in copying of the plasmid including the foreign DNA. 5. This results in the production of multiple copies of a single gene – Gene Cloning to be used to create GMOs OR This results in GMOs that produce a target protein. © 2011 Pearson Education, Inc.

Transgenic Organisms • Living things that contain recombined DNA (DNA from another organism). • After the gene has been put into the new DNA it must be placed in a living cell. • These organisms can be used in: – Medical – Pharmaceuticals – Agriculture – Environmental Solutions – Gene Therapy Examples!

Figure 20. 24

Figure 20. 2 Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Cell containing gene of interest Gene of interest 2 Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed from gene of interest Gene of interest Protein harvested Copies of gene Basic research on gene 4 Basic research and various applications Gene for pest Gene used to alter Protein dissolves resistance inserted bacteria for cleaning blood clots in heart into plants up toxic waste attack therapy Basic research on protein Human growth hormone treats stunted growth

Pope Francis gave his personal blessing to Golden Rice (GR). Why is this significant? Vitamin A deficiency (VAD) is responsible for 500, 000 cases of irreversible blindness and up to 2 million deaths each year. Particularly susceptible are pregnant women and children. Across the globe, an estimated 19 million pregnant women and 190 million children suffer from the condition. The good news, however, is that dietary supplementation of vitamin A can eliminate VAD. One way that holds particular promise is the administration via Golden Rice, which had been engineered to produce large amounts of vitamin A.

Figure 20. 23 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient 4 Inject engineered cells into patient. Bone marrow

Screening for successful gene insertion • Antibiotics kill most bacteria unless they have antibiotic resistance genes in their plasmid. • Therefore scientists can use antibiotic resistance genes as ‘markers’ to determine if their recombination and transformation was successful.

Recombinant Plasmid Lab Activity • READ ALL INSTRUCTIONS!!! • Think about what each step represents. Why would the cell/scientist complete each step? What is the source of each DNA molecule? • Be certain about your “cuts”. – Match sequences and use “sticky ends” • To Turn In: recombinant plasmid, all questions, plasmid map, data table OR detailed description of your enzyme choice • 15 minutes

Putting this to Use: 1. Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into specific types of cells, for example bone marrow • Gene therapy creates both technical and ethical questions… © 2011 Pearson Education, Inc.

Technology 3 : Full Organism Cloning Cloning • A pluripotent cell is one that can generate a complete new organism (stem cells) • Plant cloning is used extensively in agriculture © 2011 Pearson Education, Inc.

Cloning Cloning Animals: Nuclear Transplantation • In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell • Experiments with frog embryos have shown that a transplanted nucleus has a lower success rate if the donor cell is older or more differentiated. © 2011 Pearson Education, Inc.

Figure 20. 18 EXPERIMENT Frog embryo Frog egg cell Frog tadpole UV Less differentiated cell Fully differentiated (intestinal) cell Donor nucleus transplanted Enucleated egg cell Egg with donor nucleus activated to begin development RESULTS Most develop into tadpoles. Most stop developing before tadpole stage.

Reproductive Cloning of Mammals • In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell • Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus © 2011 Pearson Education, Inc.

Figure 20. 19 a TECHNIQUE Mammary cell donor 1 Cultured mammary cells Egg cell donor Egg cell from ovary 3 Cells fused 2 Nucleus removed Nucleus from mammary cell

Figure 20. 19 b Nucleus from mammary cell 4 Grown in culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor

Problems Associated with Animal Cloning • Only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects • Epigenetic changes, such as methylation of DNA, must be reversed in the donor DNA in order for genes to be expressed or repressed appropriately for early stages of development © 2011 Pearson Education, Inc.

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules • The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. © 2011 Pearson Education, Inc.

Figure 20. 8 5 TECHNIQUE 3 Target sequence Genomic DNA 1 Denaturation 3 5 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Technology #2: Gel Electrophoresis • Separating and viewing fragments of DNA Made possible because: • DNA has variations called VNTRs (variable number of tandem repeats) • Because of these different sequences restriction enzymes create RFLPs (restriction fragment length polymorphisms) • DNA has an overall negative charge. © 2011 Pearson Education, Inc.

Gel Electrophoresis • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties • A current is applied that causes charged molecules to move through the gel • Molecules are sorted into “bands” by their size © 2011 Pearson Education, Inc.

Figure 20. 9 a TECHNIQUE 1 Mixture of DNA molecules of different sizes Power source Cathode Anode Wells Gel 2 Power source Longer molecules Shorter molecules

Figure 20. 9 b RESULTS

Applications • Identification: Forensics, Missing Persons – An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids – Utilizes VNTRs and STRs – DNA testing can identify individuals with a high degree of certainty • Paternity – Offspring bands must come from parents • Evolutionary Histories/ Fossils – The more segments of DNA (bands) in common the more related • Sequencing DNA/Mapping DNA Fragments • Diagnosing Genetic Disorders/ Creating Genome Maps

Human Genome Project • In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition • Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100– 300 base pairs • SNPs can be measured by gel electrophoresis. • This information can lead to the development of gene therapies. © 2011 Pearson Education, Inc.

Figure 20. 10 Normal -globin allele 175 bp Dde. I Large fragment 201 bp Dde. I Normal Sickle-cell allele Dde. I Large fragment Sickle-cell mutant -globin allele 376 bp Dde. I Large fragment Dde. I (a) Dde. I restriction sites in normal and sickle-cell alleles of the -globin gene 201 bp 175 bp 376 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

Practice ! • © 2011 Pearson Education, Inc.

Medical: Diagnosis and Treatment of Diseases • Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation • SNPs may be associated with a disease-causing mutation • SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer © 2011 Pearson Education, Inc.

Forensic Evidence and Genetic Profiles • An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids • DNA testing can identify individuals with a high degree of certainty • Genetic profiles can be analyzed using RFLP analysis by Southern blotting © 2011 Pearson Education, Inc.

• Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences • PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths • The probability that two people who are not identical twins have the same STR markers is exceptionally small © 2011 Pearson Education, Inc.

Figure 20. 25 (a) This photo shows Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

Figure 20. 25 a (a) This photo shows Washington just before his release in 2001, after 17 years in prison.

Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials © 2011 Pearson Education, Inc.

Agricultural Applications • DNA technology is being used to improve agricultural productivity and food quality • Genetic engineering of transgenic animals speeds up the selective breeding process • Beneficial genes can be transferred between varieties or species • Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops © 2011 Pearson Education, Inc.

Figure 20. 26 TECHNIQUE Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA with the gene of interest RESULTS Recombinant Ti plasmid Plant with new trait

Safety and Ethical Questions Raised by DNA Technology • Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures • Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology © 2011 Pearson Education, Inc.

• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food • Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives • Other worries include the possibility that transgenic protein products might cause allergic reactions © 2011 Pearson Education, Inc.

• As biotechnology continues to change, so does its use in agriculture, industry, and medicine • National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology © 2011 Pearson Education, Inc.